The present invention concerns a method for examining a specimen by means of a confocal scanning microscope having at least one light source, preferably a laser, to generate an illuminating light beam for the specimen, and a beam deflection device to guide the illuminating light beam over the specimen.
The present invention further concerns a confocal scanning microscope having at least one light source, preferably a laser, to generate an illuminating light beam for a specimen, and a beam deflection device to guide the illuminating light beam over the specimen.
A method for examining a specimen by means of a scanning microscope, and a confocal scanning microscope, of the kinds cited above are known from practical use. In known scanning microscopy, a specimen is illuminated with an illuminating light beam for the specimen in order to observe the reflected or fluorescent light emitted from the specimen. The focus of the illuminating light beam is generally moved in one specimen plane by tilting two mirrors, the deflection axes usually being perpendicular to one another so that one mirror deflects in the X direction and the other in the Y direction. The tilting of the mirrors that substantially constitute the beam deflection device is brought about, for example, with the aid of galvanometer positioning elements, both fast resonant galvanometers as well as slower and more accurate non-resonant galvanometers being used. The power of the light coming from the specimen is measured as a function of the position of the scanning beam or illuminating light beam.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of an illuminating light beam. A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the light source is focused onto a pinhole, a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and detectors for detecting the detected or fluorescent light. The illuminating light or illuminating light beam must usually be coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen passes, in the most commonly used descan arrangement, via the same scanning mirrors or the same beam deflection device back to the beam splitter and passes through the latter, then being focused onto the detection pinhole behind which the detectors (usually photomultipliers) are located. Detected light that does not derive directly from the focus region takes a different light path and does not pass through the detection stop; what is obtained is a point datum that results, by way of sequential scanning of the specimen, in a three-dimensional image. A three-dimensional image is usually achieved by acquiring image data in layers.
At present, specimens are usually illuminated over the entire scan field with light of one wavelength, or simultaneously with light of several wavelengths. For this reason, comparative examinations whose purpose is to examine specimens under different spectral illumination conditions but under otherwise identical boundary conditions are performed sequentially on one specimen or sequentially on identically prepared specimens.
In cell biology, specimens are often prepared with compounds that contain calcium or amino acids such as glutamate. These xe2x80x9ccagedxe2x80x9d compounds comprise on the one hand the caged calcium or glutamate, and on the other hand the so-called complexing agents or gelators. These compounds can be broken up by irradiation with UV light or by two-photon processes; this is referred to as xe2x80x9cphotoactivation.xe2x80x9d The calcium or glutamate that is released is then capable of initiating further reactions.
Ideally, the track of the deflected illuminating light beam on the specimen surface xe2x80x94or, in the case of a confocal arrangement, in a layer plane in the specimenxe2x80x94should describe a meander. This involves first scanning a line in the X direction at a constant Y position, then a Y displacement with no change in X position, and then scanning a line in the negative X position at a constant Y position. In reality, because of the inertia of the moving galvanometer components and the mirrors of the beam deflection device, a meander shape of this kind can be approximately achieved only for low scanning rates. At reasonable scanning rates of more than 100 Hz, the scanning track of the illuminating light beam actually describes a sine-like curve, which creates the need for correction of the resulting deviations from the ideal situation. For example, the track speed in the vicinity of the reversal points is lower than in the linear sine region, resulting (inter alia) in greater bleaching in those regions. It has therefore been usual for some time to interrupt the specimen illumination while passing through the reversing portions, using mechanical stops that limit the image field or by means of suitable optical arrangementsxe2x80x94for example with acoustooptical modulators (AOTFs). This technique of interrupting the beam during scanning is called xe2x80x9cblanking.xe2x80x9d An arrangement with mechanical stops was incorporated as early as 1990 in a confocal laser scanning microscope of the applicant. An arrangement having an acoustooptical modulator is described in Scientific and Technical Information Vol. XI, No. 1, pp. 9-19, Jun. 1995, xe2x80x9cLeica TCS 4D UVxe2x80x94The system concept for Multiparameter Confocal Microscopy.xe2x80x9d This document explains the sine-like trajectory and the problems associated with it, although blanking is not explicitly mentioned. Controlled bleaching-out of any desired predefinable specimen regions using an AOTF arrangement, which makes it possible to illuminate various regions of a specimen with different light intensities, is described in P. Wedekind et al., xe2x80x9cScanning microphotolysis: a new photobleaching technique based on fast intensity modulation of a scanned laser beam and confocal imaging,xe2x80x9d Journal of Microscopy, Vol. 176, Part 1, Oct. 1994, pp. 23-33. This document illustrates a blanking technique at a very high technical level.
The German Patent Application DE 198 29 981 of Carl Zeiss Jena GmbH, xe2x80x9cMethod and arrangement for confocal microscopy,xe2x80x9d describes the elimination of the bleaching problem, and additionally the elimination of bleed-through, by the fact that the spectral composition and/or the intensity of the laser light coupled into the microscope beam path is modified while deflection continues without interruption; as a result, at least two adjacent locations or scan points of the specimen are impinged upon by light of differing spectral properties and/or different intensity.
A problem with the known method and the known confocal scanning microscope is that it is not clear how a detail of a specimen that is to be evaluated can be selected for differentiated illumination. Reliable selection and definition of the details of interest in the specimen is therefore not possible.
It is therefore the object of the present invention to make available a method for examining a specimen, wherein the method allows in a simple manner, reliable definition of details of interest of the specimen for differentiated illumination and manipulation.
According to the present invention, the aforesaid object is achieved by a method which comprises the steps of:
generating an illuminating light beam with at least one light source,
deflecting the illuminating light beam with to a beam deflection device over the specimen,
aquiring a preview image;
marking of at least one region of interest in the preview image;
allocating individual illuminating light beam wavelengths or illuminating light beam power levels to the at least one region;
illuminating the at least one region of the specimen in accordance with the allocation, wherein the illuminating light beam is guided such that substantially only the at least one marked region of the specimen is illuminated, and
performing at least one manipulation in at least one region by means of the illumination wherein during the manipulation in the at least one region that region is simultaneously observed.
It is a further object of the invention to provide a confocal scanning microscope which allows in a simple manner, reliable definition of details of interest of the specimen for differentiated illumination and manipulation. According to this, the object is accomplished by a confocal scanning microscope comprising:
at least one light source to generate an illuminating light beam;
a beam deflection device to guide the illuminating light beam over a specimen;
a least one detector for registering the reflected and fluorescent light proceeding from the specimen;
means for acquiring a preview image of at least a part of the specimen;
and
means for marking at least one region of interest in the preview image, such that individual illuminating light beam and wavelengths and illuminating light beam power levels are allocated to the at least one region and thereby performing a manipulation in the at least one region.
First a preview image is acquired. This supplies to the observer a visual depiction of the specimen being examined. Marking of at least one region of interest in the preview image is then accomplished. These two method steps make possible, in a manner according to the present invention, particularly simple selection and definition of a detail of interest of a specimen. The observer simply needs to study the preview image in order then to make a marking in the preview image.
This is then followed by an allocation of individual illuminating light beam wavelengths and/or illuminating light beam power levels to the region or regions. The region or regions of the specimen is or are then illuminated in accordance with the allocation, at least one manipulation in at least one region being performed by means of the illumination. Quite individually selected regions can thereby be subjected to a manipulation.
In an advantageous embodiment of the method, during the manipulation in at least one region, that region or those regions and/or at least one other region could be substantially simultaneously observed. This eliminates the disadvantage that between a manipulation and a sequentially occurring observation, events that are unobservable or only partly observable occur. Quasi-simultaneous and location-specific manipulation and observation are thereby made possible. In this context, it is in principle also possible to manipulate and quasi-simultaneously observe the same region.
After illumination of the region or regions of the specimen, the reflected and/or fluorescent light proceeding from the specimen could be detected. An overview image of the specimen after illumination could thereby be made available.
In an advantageous embodiment of the method, in order to avoid any undesired exposure of the specimen outside the region or regions to be examined, the illuminating light beam could be guided in such a way that substantially only the marked region or regions of the specimen are illuminated. The illuminating light beam could travel the shortest distance to the selected region or regions, or between the selected regions.
Concretely, during ongoing acquisition of an overview image of at least one region, a chemical reaction could be initiated or compounds could be broken up by controlled, preferably time-limited introduction of the illuminating light of a second light source, preferably a laser. The compounds could be caged calcium or caged glutamate compounds. The reaction of at least one other region to the breaking-up operation could be observed. The second light source could be an infrared or UV laser.
Alternatively or in addition to the triggering of a chemical reaction or the breaking up of compounds, the manipulation could comprise the excision of portions of a cell nucleus or of a complete cell nucleus. This manipulation as well could be accomplished, during ongoing acquisition of an overview image, in at least one predefinable region by controlled, preferably time-limited introduction of the illuminating light of a second light source, preferably a laser.
Portions excised in this fashion could be suctioned off with a micropipette, or transported off with a laser trap. The method suitable in this context is to be selected as required.
Concretely, the method according to the present invention allows an investigation of information transmission from cell to cell. Information transport from cell to cell takes place on the one hand by way of electrical information transfer, and on the other hand by the transmission of neurotransmitters such as, for example, calcium.
In the concrete application, a cell could be marked in the preview image. Such a cell could, for example, be prepared with a caged calcium compound.
The cell could then be illuminated at a preselected point in time with UV or infrared light. The illumination is performed, in this context, in controlled and location-specific fashion.
The aforesaid caged calcium compound could thereby be broken up, and the calcium that is released could initiate a reaction in the cell. Information transmittal could be detected by observation of an adjacent cell. For that purpose, concretely, the adjacent cell could be prepared with a calcium indicator.
It is known that the reaction of the adjacent cell can fail to occur if the stimulus information of a third cell arrives within a specific time window. In particular for the investigation of this phenomenology, it may be useful to initiate a reaction in two cells quasi-simultaneously or with a defined time offset. This, too, is made possible by the method according to the present invention. It is furthermore conceivable to alternately illuminate different scan points in the two cells.
In both a two-dimensional X-Y depiction and in a three-dimensional X-Y-Z depiction, the region or regions of interest of the specimen could be selected or marked by way of a computer and preferably a computer mouse.
The allocation of individual illuminating light beam wavelengths and/or illuminating light beam power levels to the region or regions could then also be accomplished by way of a computer of this kind.
To prevent illumination of the specimen outside the region or regions, a definable blanking could be performed. In this, the illuminating light beam is interrupted in controlled fashion during scanning, so that the unmarked regions are not illuminated at all. As a result, the region or regions is or are particularly emphasized, and the unmarked remaining region of the specimen is not unnecessarily bleached out.
To achieve higher contrast and to decrease the total data acquisition time, the region or regions could be scanned more slowly and with elevated photon statistics as compared to the remainder of the specimen.
Outside the region or regions or between the regions, the specimen could be scanned at the maximum deflection speed. A further reduction in total data acquisition time could be achieved by the fact that outside the region or regions or between the regions, beam deflection deviates from sinusoidal, sawtooth-shaped, or meander-shaped beam deflection. The regions could thereby be arrived at over a shorter distance. Ideally, beam deflection between two regions or the regions could be accomplished substantially in a direct line from one region to another region.
In an advantageous embodiment, during manipulation in at least one region, that region or those regions and/or at least one other region could be substantially simultaneously observable.
Alternatively or in addition thereto, after illumination of the region or regions of the specimen, the reflected and/or fluorescent light proceeding from the specimen could be detectable.
Concretely, the confocal scanning microscope could comprise a spectrally selective element for setting the illuminating light beam wavelength or wavelengths. The spectrally selective element could be an AOTF (acoustooptical tunable filter), an AOD (acoustooptical deflector), an EOM (electrooptical modulator), or a mechanical component. Acoustooptical tunable filters are characterized by great flexibility, and make it possible to switch over the illuminating light beam wavelengths, introduce light of one or more wavelengths, or vary the light power level, very quickly, i.e. in the range of approx. 1 xcexcs or less.
A spectrally selective element of this kind could be controllable by way of a computer, preferably as a function of the deflection position.
The confocal scanning microscope could furthermore comprise an element for adjusting the illuminating light beam power level. An element of this kind for adjusting the illuminating light beam power level could comprise an AOTF or a mechanical component. The element for adjusting the illuminating light beam power level could also be controllable by way of a computer, preferably as a function of the deflection position.
In particularly simple fashion, the same element could be usable for adjusting the illuminating light beam wavelength or wavelengths and for adjusting the illuminating light beam power level. An AOTF is particularly suitable in this context.
To make available several different illuminating light beam wavelengths, several lasers could be provided to generate the illuminating light beam. Alternatively, one or more multiple-line lasers could also be provided to generate the illuminating light beam.
A PC, on whose monitor the image or preview image of the specimen is displayed, could be usable for displaying and marking the region or regions.
The marking of a three-dimensional region or regions could be performable in an X, Y, Z depiction or in two-dimensional sectioned depictions.
In particularly simple fashion, the beam deflection device could comprise galvanometer positioning elements. Galvanometer positioning elements of this kind could preferably be controllable by way of a computer, with which the beam deflection speeds can be adapted individually to requirements in terms of the marked region or regions.
Blue semiconductor lasers or Ar lasers are preferably used for UV illumination. Ti:sapphire lasers preferably provide illumination in the infrared region.